Therapeutic implications for localized prostate cancer by multiomics analyses of the ageing microenvironment landscape

Background: Numerous studies have substantiated the association between aging and the progression of malignant tumors in humans, notably prostate cancer (PCa). Nevertheless, to the best of our knowledge, no studies have comprehensively elucidated the intricate characteristics of the aging microenvironment (AME) in PCa. Methods: AME regulatory patterns were determined using the NMF algorithm. Then an ageing microenvironment index (AMI) was constructed, with excellent prognostic and immunotherapy prediction ability, and its' clinical relevance was surveyed through spatial transcriptomics. Further, the drug response was analysed using the Genomics of Drug Sensitivity in Cancer (GDSC), the Connectivity Map (CMap) and CellMiner database for patients with PCa. Finally, the AME was studied using in vitro and vivo experiments. Results: Three different AME regulatory patterns were identified across 813 PCa patients, associated with distinct clinical prognosis and physiological pathways. Based on the AMI, patients with PCa were divided into the high-score and low-score subsets. Higher AMI score was significantly infiltrated with more immune cells, higher rate of biochemical recurrence (BCR) and worse response to immunotherapy, antiandrogen therapy and chemotherapy in PCa. In addition, we found that the combination of bicalutamide and embelin was capable of suppressing tumor growth of PCa. Besides, as the main components of AMI, COL1A1 and BGLAP act as oncogenes and were verified via in vivo and in vitro experiments. Conclusions: AME regulation is significantly associated with the diversity and complexity of TME. Quantitative evaluation of the AME regulatory patterns may provide promising novel molecular markers for individualised therapy in PCa.


Cell viability and apoptosis assay
The cell proliferation rate was assessed by Cell Counting Kit-8 (CCK-8; Dojindo, Japan) every 24h through absorbance measurement at 450 nm using a plate reader (VARIOSKAN LUX, Thermo Fisher Scientific, USA). Flow cytometry was applied to evaluate apoptosis. After being washed twice with cold PBS, cells were resuspended in the Annexin V binding buffer at a concentration of 10 6 cells/mL and cultivated with AlexaFluor 647 Annexin V (Biolegend, USA) at 4 °C for 15 min away from light.
Subsequently, cells were added with PI (Sigma, USA) and then immediately analysed via flow cytometry (FACSCanto II, BD, USA).

Immunofluorescence microscopy
To detect the protein expression of two AME regulators (COL1A1 and BGLAP) before and after knockdown, PC3 and DU145 cells were fixed, permeabilised and prehybridised.
Subsequently, the cells were incubated in the blocking buffer (PBST with 5% bovine serum albumin) at room temperature (RT) for 30 min, primary antibodies (1:200 dilution) at RT for 1 h and then secondary antibodies conjugated with Alexa Fluor 488-or 594-(1:200 dilution, Cell Signaling Technology) at RT for 1 h away from light, followed by incubation with DAPI (Vector Laboratories) for 10 min. The immunofluorescence images were generated using the confocal microscope. Antibody information is listed in Supplementary Table S4.

Immunohistochemical analysis
PCa tumor tissues and adjacent normal prostate tissues from 78 patients performed radical prostatectomy at the First Affiliated Hospital, Sun Yat-sen University, were examined via immunohistochemical (IHC) analysis. Tissue sections were incubated with anti-COL1A1 and anti-BGLAP (1:100 dilution), and the staining intensity was estimated using a histologic score (H-score) system via digital pathology image analysis. The H-score of each sample ranged from 0 (no staining) to 300 (maximum immunoreactivity) and was calculated based on immunostaining intensity and the corresponding percentage.
In particular, the staining intensity was assessed by the ranking from 0 to 3, with 0, 1, 2, 3 representing negative staining, weak staining, moderate staining and strong staining respectively. Thereafter, the H-score was calculated according to the following formula: H-score = 3 × (% at 3) + 2 × (% at 2) + 1 × (% at 1). The experiments were reviewed and approved by the Ethics Committee of the First Affiliated Hospital, Sun Yat-sen University (GZJZ-SB2020-027). Written informed consent were provided by all patients participating in this study.

Colony formation assay
1×10 3 PCa cells in the logarithmic growth phase were seeded into 6-well plates and cultured for 7-10 days. When the number of cells in most single clones was over 50, cells were washed with PBS (dissolved in methanol), fixed with 4% paraformaldehyde for about 30 mins and stained by 0.1% crystal violet, then the number of colonies was quantified.

Cell migration assay
Cell migration assay was conducted using a transwell system with a 24-well inserted plate (5.0-μm pore size) following the manufacturer's instructions. The suspension of PC3 and DU-145 cell lines (1 × 10 5 cells/well) in 200 μL of FBS-free RMPI-1640 was added to the upper chamber. Simultaneously, 800 μL of RMPI-1640 (with 20% FBS) was added to the lower chamber. After being incubated for 24 h, cells attached to the lower chamber were fixed and then stained with 0.1% crystal violet for about 20 min.

In vivo animal experiments
All animal experiments were approved by the Institutional Ethics Committee of the First Affiliated Hospital, Sun Yat-sen University. To assess the efficacy of bicalutamide-loaded micelles in vivo, three million LNCaP cells suspended in 1:1 media and matrigel were injected subcutaneously to 6-week old male BALB/C nude mice to induce xenograft flank tumors. When tumors grew to about 150 mm 3 , mice were classified into three groups of five mice randomly to reduce differences in tumor size and weight. Mice in the three groups were injected intratumorally with saline, sonicated bicalutamide suspension and bicalutamide-loaded micelles (20 mg/kg) respectively three times a week. The sizes of tumors were measured by a caliper before each injection, and tumor volumes were calculated with the formula: (width 2 × length)/2.
To examine the therapeutic potential of targeting COL1A1 and BGLAP in patients with PCa, an in vivo lung colonisation assay was performed. First, stable cell lines (PC3) with COL1A1 or BGLAP knockdown were established. Subsequently, 20 mice were split into four groups (COL1A1-NC, COL1A1-SH, BGLAP-NC and BGLAP-SH; n = 5), and 2 × 10 6 PC3 cells were transfected with the corresponding plasmid via tail-vein injection.
After approximately 6-8 weeks, the mice were sacrificed using CO2 as an anaesthetic, and their lungs were removed. The lung tissues were soaked in picric acid and embedded in paraffin for H&E staining. Representative nodule images were captured after H&E staining. We also establish the orthotopic-xenograft prostate-tumor mouse models to further survey the effect of promoting tumor growth.  The correlation among AME regulators in prostate cancer. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.